KR20160131238A - Novel strain having C4 and C6 organic acid producing capacity, and method for producing bio-fuel using the strain - Google Patents

Abstract

The present invention relates to a novel strain having ability to produce C4 and C6 organic acids and a method for producing a biofuel using the novel strain. More specifically, the present invention relates to a method for producing a biofuel using C4 Producense galactitol riborans ( Caproiciproducens galactitolivorans and a method for producing biofuel using the same.
According to the present invention, there is provided a novel strain which simultaneously produces C4 and C6 organic acids with excellent productivity when cultivated in a medium containing a carbon source derived from various biomass or organic waste resources, C8-C12 compounds, which are high-energy compounds, and biofuels.

Description

[Technical Field] The present invention relates to a novel strain having C4 and C6 organic acid producing ability and a method for producing biofuel using the novel strain,

The present invention relates to a novel strain having ability to produce C4 and C6 organic acids, and a method for producing a biofuel using the novel strain.

80% of the energy used on the earth is produced from fossil fuels, and most of the raw materials such as plastic raw materials, synthetic rubber, solvents, paints and adhesives are produced from petrochemical processes. Therefore, it is necessary to continuously supply fossil fuels, but due to the limitation of the reserves, it is urgent to develop new alternative fuels. At present, various candidate substances such as biodiesel, alcohol and biomass have been proposed as alternative fuels. However, there have been proposed methods of converting biomass obtained from nature into alternative fuel by controlling pressure, catalyst, Has also been proposed, but it shows limitations in production volume and efficiency.

Carbohydrates contained in biomass can be converted into various biofuels and biochemical raw materials such as alcohols, carboxylic acids, ethers, esters, etc. Especially, the saccharides can be converted into alcohols and C3-C6 Can be converted to important basic chemicals including carboxylic acids. Research on the production and use of ethanol as a biofuel derived from renewable vegetable raw materials has been advanced, but the research on producing C4 material such as butanol or C6 material, such as hexanol, is still at an early stage.

For example, prior art patents for improving the production yield of bioethanol and the like are disclosed in Korean Patent Laid-Open Nos. 10-2014-0050226, 10-2012-0038782, 10-2012-0082141, 10-2012 -0082138 and 10-2011-0007981, but the number of patent documents relating to biobutanol or biohexanol is relatively small. Korean Patent Laid-Open Publication No. 10-2012-0037217 discloses a method for increasing the production yield by adding a gene level manipulation to an enzyme involved in the production route of biobutanol.

On the other hand, it is well known that n-butanol and n-butyric acid which are representative C4 materials are produced by performing acetone-butanol-ethanol fermentation (ABE fermentation) mainly by anaerobic bacteria in Clostridium (BH Kim and GM Gadd, Bacterial physiology and metabolism. Cambridge University Press, Cambridge (2008)), for example, Clostridium beijerinckii and Clostridium acetobutylicum are well known. In addition, microorganisms producing n-hexanoic acid, which is a representative C6 material, include Clostridium kluyverii , Megasphaera < RTI ID = 0.0 > elsdenii ).

However, no microorganisms having the ability to produce C4 organic acid and C6 organic acid at the same time in the carbon source medium have been reported, and furthermore, studies on producing biofuels including C4 alcohol and C6 alcohol have been reported none.

Korean Patent Publication No. 10-2014-0050226 Korean Patent Publication No. 10-2012-0038782 Korean Patent Publication No. 10-2012-0082141 Korean Patent Publication No. 10-2012-0082138 Korean Patent Publication No. 10-2011-0007981 Korean Patent Publication No. 10-2012-0037217

B. H. Kim and G. M. Gadd, Bacterial physiology and metabolism. Cambridge University Press, Cambridge (2008)

Accordingly, the present invention provides a novel strain capable of producing C4 organic acid and C6 organic acid with excellent production ability when cultivated in a medium containing a carbon source, and a method for producing a biofuel using the strain.

In order to solve the above problems of the present invention,

Carbon source medium from kapeuroyi Procedures Two sense galactose Tito with simultaneous production capacity of C4 C6 organic acids and organic acid when culturing provides a ribonucleic lance (Caproiciproducens galactitolivorans) strain (KCCM10991P).

According to an embodiment of the present invention, the carbon source medium may include at least one saccharide selected from the group consisting of glycerol, glucose, galactitol, mannose, and fructose.

According to another embodiment of the present invention, the C4 organic acid may be butyric acid and the C6 organic acid may be hexanoic acid.

Further, according to the present invention,

a) culturing a strain of caproicprodense galactibolans in a carbon source medium to produce a C4 organic acid and a C6 organic acid;

b) separating the C4 organic acid and C6 organic acid from the product of step a);

c) adding C4 alcohols and C6 alcohols having the same mole number as the C4 organic acids and C6 organic acids separated from step b) to prepare a mixture;

d) performing a catalytic reaction on the mixed solution of step c) to prepare a C8-C12 compound; And

e) producing a C4 alcohol and a C6 alcohol by reacting the C8-C12 compound prepared from step d) with hydrogen gas

And a method for producing the biofuel.

According to an embodiment of the present invention, the carbon source medium may include at least one saccharide selected from the group consisting of glycerol, glucose, galactitol, mannose, and fructose.

According to another embodiment of the present invention, the C4 organic acid may be butyric acid and the C6 organic acid may be hexanoic acid.

According to another embodiment of the present invention, the cultivation of step a) may be carried out by anaerobic culture at 35 ° C to 45 ° C for 24 hours to 72 hours.

According to another embodiment of the present invention, the culture of step a) may be carried out in a culture medium containing galactitol, fructose, mannose, tagatose, glucose and glycerol.

According to another embodiment of the present invention, the separation in step b) may be carried out using an ion exchange resin, or may be carried out by extracting the C4 organic acid and C6 organic acid from the culture medium using an organic solvent which is immiscible with water have.

According to another embodiment of the present invention, the C4 alcohol may be butanol, and the C6 alcohol may be hexanol.

According to another embodiment of the present invention, the catalytic reaction of step d) may be carried out in the presence of a catalyst selected from the group consisting of zeolite, heteropoly acids, silica-alumina, Nafion-H, para-toluenesulfonic acid (p-toluenesulfonic acid), SO 4 ^2- / ZrO ₂ or SO 4 ^{2 -} / TiO ₂ -La ₂ O ₃ .

According to another embodiment of the present invention, the catalytic reaction of step d) may be carried out by adding at least one enzyme selected from the group consisting of esterase and lipase.

According to another embodiment of the present invention, the hydrogen gas in step e) may be hydrogen gas generated from step a).

According to another embodiment of the present invention, the carbon source of step a) may be prepared by physically crushing, washing and hydrolyzing biomass or organic waste.

According to the present invention, there is provided a novel strain which simultaneously produces C4 and C6 organic acids with excellent productivity when cultivated in a medium containing a carbon source derived from various biomass or organic waste resources, C8-C12 compounds, which are high-energy compounds, and biofuels.

1 is a graph showing the results of gas chromatography analysis of a hexanoic acid standard material.
2 is a graph showing the result of gas chromatography analysis of the culture solution of caproic acid produsense galactitol riborans.
FIGS. 3A to 3D are graphs (3a and 3b) showing the results of GC-TOF-MS analysis of the culture solution of caproicilprodosense galactitol riborans extracted with hexane and ethyl acetate as solvents, (3c and 3d), respectively.
FIG. 4 is a diagram showing a schematic diagram of caproicryptosidase galactitol riborans and related bacteria according to the present invention. FIG.
FIG. 5 is a graph showing that the extraction efficiency of organic acid is changed according to the CO ₂ pressure applied during the extraction of organic acid from the culture liquid.
6a and 6b are photographs showing the organic acid extracting apparatus used in the method according to the present invention, wherein the change in extraction amount (6a) according to the mixing rate of the culture liquid and the change in oil separation (6b) according to the stagnation time are respectively compared.
FIG. 7 is a graph showing the extraction efficiency of organic acid extracted at a pH of 6 under a pressure of 10 bar of CO ₂ while varying the ratio of the extraction solvent to the medium.
FIG. 8 is a photograph showing that gel state components are converted to a liquid state by treatment with sulfuric acid.
FIG. 9 is a graph showing the degree of microbial growth and glucose consumption according to the culturing time when the caproicryptosidase galactitol riborans according to the present invention is cultured in the medium supplemented with glucose. FIG.
FIG. 10 is a graph showing production yields of acetic acid, butyric acid and hexanoic acid according to the culture time when the caproicprodusense galactivolans according to the present invention is cultured in a medium supplemented with glucose. FIG.
FIG. 11 is a graph showing the consumption of galactitol and the production yields of acetic acid, butyric acid and hexanoic acid according to the culturing time when culturing the culture medium containing galactitol in accordance with the present invention.

Hereinafter, the present invention will be described in more detail with reference to the drawings and examples.

In the present invention, Caproicycloviridae ( Caproiciproducens galactitolivorans ) strain (KCCM 10991P) having the ability to simultaneously produce C4 organic acid and C6 organic acid when cultured in a carbon source medium is provided.

As described in detail in the following examples, in the present invention, strains producing C4 and C6 organic acids were selected using a selective medium, and products produced by the strain culture were analyzed by chromatographic analysis to obtain C4 and C6 The presence of organic acids was confirmed. Next, the selected strains were identified by the multiphasic analysis method, and it was confirmed that the selected strain was a caproiciprocepsidase galactitol riborans strain in clostridium.

The carbon source medium for culturing the strain according to the present invention includes, but is not limited to, at least one saccharide selected from the group consisting of glycerol, glucose, galactitol, mannose, and fructose, In particular, butyric acid and hexanoic acid.

According to another aspect of the present invention, there is provided a method for producing a biofuel using the strain,

a) culturing a strain of caproicprodense galactibolans in a carbon source medium to produce a C4 organic acid and a C6 organic acid;

b) separating the C4 organic acid and C6 organic acid from the product of step a);

c) adding C4 alcohols and C6 alcohols having the same mole number as the C4 organic acids and C6 organic acids separated from step b) to prepare a mixture;

d) performing a catalytic reaction on the mixed solution of step c) to prepare a C8-C12 compound; And

e) producing a C4 alcohol and a C6 alcohol by reacting the C8-C12 compound prepared from step d) with hydrogen gas

.

The culturing of the strain of step a) may be carried out by anaerobic culture at 35 ° C to 45 ° C for 24 hours to 72 hours.

According to another embodiment of the present invention, the culture of step a) may be carried out in a modified CAB culture medium containing galactitol, fructose, mannose, tagatose, glucose and glycerol. The above-mentioned Caproi producense galactivolans strain was collected from a sewage treatment plant and then sludge was heat-treated to kill microorganisms other than spore-forming microorganisms. The microorganisms were added at a predetermined volume relative to the medium and then cultured under agitation to obtain C4 and C6 It can be obtained by selecting strains which produce the maximum amount of organic acid. Only the thus selected strains can be cultured in the above-mentioned saccharide-containing culture medium to produce C4 organic acid and C6 organic acid.

On the other hand, the carbon source necessary for culturing the strain may be one prepared by pretreatment such as physically pulverizing, washing and hydrolyzing biomass or organic waste resources. The pretreatment of the organic waste may improve the efficiency of dissolving organic components Maximize the biohydrogen production time, minimize the biological hydrogen production time by anaerobic fermentation, and increase the efficiency thereof. Therefore, by physically pulverizing biomass or organic waste resources, the microorganism is made to have a size suitable for use as a substrate, and a component that shows toxicity to microorganisms such as chlorine ions is removed through a washing process, There is a need to improve the solubility of the organic component by subjecting it to a decomposition process.

Then, the step of separating only the produced C4 and C6 organic acids should be performed. The separation in step b) may be carried out using an ion exchange resin, or may be carried out using an organic solvent which is immiscible with water, From the culture broth. For example, when an organic acid is separated using an ion exchange resin, a C4 organic acid such as butyric acid and a C6 organic acid such as hexanoic acid show a negative charge in the culture liquid. Therefore, when an anion exchange resin is used, only organic acid components Can be selectively adsorbed. In addition, the adsorbed organic acids can be desorbed from the anion exchange resin using an alkali solution, an acid solution, ethanol, or the like. When an organic solvent for extraction is used to extract an organic acid from a culture solution, an organic solvent such as butyl butyrate, dodecanol, oleyl alcohol, etc. may be mixed with the culture solution, and then the solution may be stirred and extracted. An organic solvent containing an organic acid and a culture solution obtained by extracting an organic acid are separated using an oil-water separator or the like, and the organic acid can be separated by performing an additional chemical reaction with the separated organic acid-containing organic solvent.

In the next step, various C8-C12 compounds can be prepared by carrying out an esterification reaction on the produced C4 organic acid and C6 organic acid. For example, the reactions for preparing various C8-C12 compounds from hexanoic acid and butyric acid by an esterification reaction using an acid catalyst and an esterase are shown in the following Schemes 1 to 4.

<Reaction Scheme 1>

<Reaction Scheme 2>

<Reaction Scheme 3>

<Reaction Scheme 4>

The chemical catalysts and enzymes involved in the reactions of the above Schemes 1 to 4 include, but are not limited to, zeolite, heteropoly acids, silica-alumina, Nafion-H , P-toluenesulfonic acid, SO4 ^{2 -} / ZrO ₂ Or SO4 ^{₂ -} / TiO ² -La may be at least one enzyme selected from the superacid chemistry catalyst, esterase or lipase, and the group consisting of such as ₂ O ₃ as an example. In particular, when an esterase enzyme is used, it can be easily recovered and economically reused when the enzyme is immobilized on a carrier such as nanofiber, carrageenan, gelatin or beads.

Then, the hydrogenation reaction of the C8-C12 ester compounds prepared as described above enables various alcohol compounds to be prepared. For example, in the following Reaction Schemes 5 to 8, various alcohol compounds are produced by the hydrogenation reaction of the ester compounds formed by the above-described Reaction Schemes 1 to 4.

<Reaction Scheme 5>

<Reaction Scheme 6>

<Reaction Scheme 7>

<Reaction Scheme 8>

At this time, the hydrogen gas required for the reaction may be the hydrogen gas produced by the cultivation of the strain of step a), together with the C4 organic acid and the C6 organic acid.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to assist the understanding of the present invention and should not be construed as limiting the scope of the present invention.

Example  One. C4  Organic acids and C6  Selection of strains capable of simultaneous production of organic acids

Example  1.1 C4 - C6  Isolation of organic acid-producing strains

The process of selecting microorganisms producing C4-C6 raw materials hexanoic acid and butyric acid using sugar alcohols was performed as follows.

A modified medium supplemented with galactitol in modified Clostridium acetobutyricum medium (mCAB) was used. The selective medium was prepared by adding 10 g of carboltitol, 4 g of yeast extract, 1 g of tryptone, 0.5 g of asparagine, 0.1 g of MgSO ₄ .7H ₂ O , 0.1 g of MnSO ₄ .H ₂ O, 0.015 g of FeSO ₄ .7H ₂ O, 0.1 g of NaCl, 1.5 g of KH ₂ PO ₄ and 1.5 g of K ₂ HPO ₄ were dissolved and placed in a serum bottle And then used as an anaerobic condition through argon gas replacement. The sludge used as seeds was collected from the municipal sewage treatment plant. The sludge was heat-treated at 100 ° C. for 30 minutes to kill the microorganisms other than the spore-forming microorganisms. The microorganisms were inoculated into the medium at a rate of 5% (v / v) Lt; / RTI &gt; In the cultivation process, acetic acid (C2), propionic acid (C3), butyric acid (C4), pentanoic acid (C5), hexanoic acid Organic acid and hydrogen were produced. Among the resulting organic acids, microorganisms that produce the highest amounts of hexanoic acid and butyric acid are selected, and the microorganism caproicci produsense galactitol ribrans ( Caproiciproducens galactitolivorans ) (deposited at the Korean Microorganism Conservation Center, Accession No. KCCM 10991P).

Example  1.2 In the microbial culture Hexanoic acid  Confirm creation

The C4-C6 raw material contained in the culture broth cultivated in Example 1.1 was analyzed by gas chromatography (GC-FID, agilent 6890N) equipped with a flame ionization detector (FID) Were analyzed by gas chromatography equipped with a thermal conductivity detector (TCD). In addition, the presence of hexanoic acid was confirmed using GC-TOF-MS. For the hexanoic acid separation of the culture, an organic solvent extraction method was used as described below. First, the culture liquid was dispensed into a separating funnel in the same amount as the extraction solvent, which is a mixture of hexane and ethyl acetate, and shaken strongly for 20 minutes. Then, the organic solvent layer was separated and analyzed by gas chromatography . In the GC-FID analysis, it was confirmed that the hexanoic acid standard substance was detected after about 14.0 minutes. As a result of injecting the culture solution into the same apparatus, it was confirmed that the hexanoic acid was extracted after about 14.0 minutes at the same time 1 and 2). For more accurate analysis, the presence of hexanoic acid was confirmed by comparing GC-TOF-MS with hexane and ethyl acetate extracted solvents as shown in FIGS.

Example  1.3 Microorganism Caproicin Producense Galactitol riborans of  Sympathy

To identify the novel microorganism caproicryptosidase galactitol riborans, a multi - faceted assay was applied. For physiological and biochemical characterization, API 20NE and API 50CH were examined and fatty acid composition was analyzed, and 16S ribosomal DNA was analyzed for gene analysis.

API inspection was performed by API standard method. In the API 20A test, caproic acid produsense galactitol riborans was negative in the case of indole production, urease, and citric acid utilization, and the gelatin hydrolysis reaction was positive. API 50CH, which is a test for confirming the use of 49 kinds of carbon sources, can be used in the case of glycerol, D-arabinose, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, dapsitol, inositol, N-acetylglucosamine, cellobiose, maltose, lactose, starch, glycogen, D-tagatose and L-fucose were used. When 0.1 M each of mannitol, mannose, lactose, glucose, D, L-arabinose, fructose, and xylose was added to a medium containing 0.5 g / L of Na ₂ S 9H ₂ O as a reducing agent Each of these carbon sources was used as a substrate, and hexanoic acid was produced at about 1 g / L.

FAMEs (Fatty Acid Methyl Esters) analysis was performed to analyze the membrane fatty acid composition of the microorganism caproic acid produsense galactitol riborans. The caproic acid produsense galactolytoliborance cultures were recovered and hydrolyzed with a sodium hydroxide / methanol solution to separate the fatty acids, and then formed into a femme with hydrochloric acid / methanol solution. Femmes were extracted with organic solvents and analyzed by gas chromatography. % Composition of the Clostridium genus kapeuroyi Procedures Two sense galactose membrane fatty acid component of Tito ribonucleic lance is _{C 14: 0 (3.17%)} , C 16: 0 (3.12%), C 14: 0 DMA (5.41% _{), C 16: 0 DMA (} 22.15%), C 18: 0 DMA (4.04%), C 16: 0 ALDE (14.09%), C 18: 0 ALDE (3.13%), anteiso-C 17: 0 FAME ( _{5.99%), C 18: 1} CIS 9 (1.12%), C 18: 1 CIS 9 DMA (7.65%), C 18: 1 CIS 11 DMA (4.62%), C 18: 2 CIS 9, 12 (1.08% ), Summed features 1 (2.52%), Summed features 5 (2.84%); Summed features 7 (5.56%), Summed features 8 (3.49%) and Summed features 11 (10.03%).

For 16S ribosomal DNA analysis of the novel microorganism caproicryptosyltransferase of the present invention, 27S and 1492R, which are primers capable of amplifying 16S ribosomal DNA and extracting DNA from the culture, DNA was amplified with primers to obtain a PCR product of about 1.3 Kb. The sequence of this DNA fragment was analyzed to confirm the nucleotide sequence of about 1259 bp (SEQ ID NO: 1). The nucleotide sequence of the 16S ribosomal DNA of Caproicus prodensense galactitol riborans was compared with the 16S ribosomal DNA sequence of a known bacterium. Sequence analysis revealed that caproiciproceans galactitol riborans was identified as a clostridium microorganism. Caprocyte Producense The most closely related microorganism to galactitol riborans is Clostridium spp. sporosphaeroides) DSM 1294 ^T and repteom Clostridium (Clostridium leptum ) DSM 753 ^T , showing 94.49% and 94.26% 16S ribosomal DNA homology, respectively.

In addition, for the phylogenetic analysis, the 16S ribosomal DNA sequence of Caproicus prodensense galactitol riborans was compared with the sequence of the microorganisms. The sequences of the microorganisms were sorted using the cluster X program and the sorted sequences were analyzed using the MEGA3 program. In the scheme made by the neighbor-joining method, the caproic acid produsense galactitol riborans belonged to the same group as Clostridium spores ferroide DSM 1294 ^T (see FIG. 4).

Therefore, through analysis of 16S ribosomal DNA homology and phylogenetic analysis, caproic acid produsense galactitol riborans was identified as Clostridium as a new microorganism which was previously unknown. Clostridium spores Feroids DSM 1294 ^T and Clostridium leptom DSM 753 ^{T, which} are closest to the caproic acid produsense galactitol riborans, produced acetic acid and butyric acid during the culture but did not produce hexanoic acid Respectively.

Example  2. Caproic acid Producense Galactitol riborans on  Butyric acid and Hexa No Iksan production

Caprocyte Producense The optimum conditions for the production of hexanoic acid from galactitol riborans was that a rich complex nitrogen source was required and at the same time a low redox potential was required. As a complex nitrogen source, beef extract, yeast extract, tryptone, and peptone were used. In order to keep the redox potential low, Na ₂ S 5H ₂ O to 9H ₂ O or Cystein HCl was added as a reducing agent. Addition of 0.5 g / L or more of Na ₂ S 5 H ₂ O to 9H ₂ O or cysteine HCl to the culture medium results in toxicity to caproic acid produsense galactitol riborans. And the reducing power was increased by mixing the two reducing agents to the extent that the reducing agents did not show any toxicity. When cysteine-HCl was added to the medium, an oxidation-reduction potential of about -100 mV was obtained, and when Na ₂ S 5H ₂ O to 9H ₂ O was added, an oxidation-reduction potential of about -500 mV was obtained I could.

The composition of the culture medium suitable for culturing caproiciprogesophage galactitol riborans was described below.

Caprocyte prodysense galactolytolibolance was added to a hexanoic acid production medium 1 (composition: trypticase peptone 5.00 g / L, peptone 5.00 g / L, yeast extract 10.00 g / L, beef jam 5.00 g / g / L carbon source and 0.50 g / L of heptane ssiseu -HCl a reducing agent, a mineral element _{CaCl 2 × 2H 2 O 0.25 g} / L, MgSO 4 × 7H 2 O 0.50 g / L, K 2 HPO 4 1.00 g / L , KH ₂ PO ₄ 1.00 g / L, and NaHCO ₃ 10.00 g / L) at 120 rpm in a shaking incubator set at an initial pH of 7.2 to 40 ° C. As a result of observation for about 2 days, caproic acid produsense galactitol riborans produced hexanoic acid and butyric acid using glucose as a substrate. The turbidity was measured using a spectrophotometer, and as a result, Two senses galactitol riborans grew. The production of hexanoic acid was about 1.5 g / L and the production of butyric acid was about 0.7 g / L. Production medium 2 (Composition: 1 L of distilled water was mixed with 4 g of carbolititol, 5 g of yeast extract, 5 g of K ₂ HPO ₄ , 4 g of glucose, 1 g of maltose, 1 g of cellobiose, Soluble starch, 125 g of meat-cooked medium, 0.25 g of cysteine) and production medium 3 (composition: 1 L of distilled water, 3 g yeast extract, 10 g beef essence, 20 g galactitol, 5 g glucose , 5 g of tryptone, 1 g of soluble starch, 1 g of sodium acetate, 0.25 g of cysteine). As a result, caproic acid produsense galactitol riborans produced hexanoic acid, butyric acid, and acetic acid (see FIG. 2). After 5 days of culture without pH adjustment of the culture medium in the same medium, the concentration of caproic acid prodense galactitol riborans was increased to OD ₆₀₀ 2.3. As the main metabolite, 2 to 3 g / L of hexanoic acid And about 2 g / L of butyric acid were produced.

FIG. 9 is a graph showing the degree of microbial growth and glucose consumption according to the culture time when the caproicryptosus galactitol riborans according to the present invention was cultured in the medium supplemented with glucose, and FIG. The production amount of acetic acid, butyric acid, and hexanoic acid is shown in the graph. FIG. 11 shows the results of cultivating caproicprodense galactobilis according to the present invention in the medium supplemented with galactitol, The yields of acetic acid, butyric acid and hexanoic acid are shown graphically.

Example  3. The produced butyric acid and Hexanoic acid  collection

The separation of butyric acid and hexanoic acid produced by caproic acid produsense galactitol riborans was carried out by extraction. Extraction was carried out using an organic solvent such as hexane, ethyl acetate, diethyl ether, chloroform and the like which is immiscible with water. The culture solution and the organic solvent immiscible with water were mixed and allowed to stand for several minutes in the same volume, and then butyric acid and hexanoic acid were extracted from the culture product of caproicosylprototolibolance. In addition, the pH of the culture broth was lowered to about 3 using nitric acid before extraction, and the extraction efficiency was increased by maintaining the pKa value or less for each acid. As in the apparatus shown in FIG. 6, extraction was performed using a high-pressure reactor capable of lowering the pH without adding strong acid for rapid extraction, and the extraction efficiency was increased by pressurizing CO ₂ to about 10 to 50 bar. When the pressure of CO ₂ in the solution is formed into a bicarbonate, such as CO ₂ to the Scheme 9 and the pH is temporarily reduced, thereby increasing the extraction efficiency of the organic acid.

<Reaction Scheme 9>

CO ₂ + H ₂ O → H ₂ CO ₃ → H ⁺ + CO ₃ ^-

FIG. 5 is a graph showing that the extraction efficiency of organic acid is changed according to the applied CO ₂ pressure. Referring to FIG. 5, it can be seen that the extraction efficiency increases as the CO ₂ pressure increases. FIG. 7 shows the extraction efficiency of the extracted organic acid by varying the ratio of the extraction solvent to the medium while applying a pressure of 10 bar of CO ₂ at a pH of 6. Referring to FIG. 7, it was observed that the extraction efficiency of the organic acid was changed depending on the volume of the extractant, and the extraction efficiency was increased as the volume of the extraction solvent was increased.

In addition to using an organic solvent, butyric acid and hexanoic acid were extracted from the culture solution using a primary alcohol compound. Examples of materials that can be used as the primary alcohol or other extraction solvent include oleyl alcohol, dodecanol, decanol, MIBK, kerosene, liquid paraffin, etc. In particular, oleyl alcohol has low solubility in water, While maintaining the activity of the enzyme. Furthermore, extraction of butyric acid and hexanoic acid was possible as a tertiary amine, and it was possible to extract de-tridecylamine, alamine 336 (alamine 336, tri-N-octylamine), aliquot, And so on. In addition, a mixture of tertiary amine and primary alcohol in a certain ratio (1: 9 to 2: 8) was added to the culture solution to separate butyric acid and hexanoic acid continuously in the bi-phase state.

As an alternative method of organic acid separation without using organic solvent, there was an anion exchange resin method. Since hexanoic acid showed a negative charge in the culture solution, hexanoic acid could be adsorbed in the culture liquid using an anion exchange resin, The hexanoic acid could be desorbed from the anion exchange resin using alkali solution, acid solution and ethanol. The anion exchange resins used were reusable. 5 shows the results of extracting butyric acid produced by the microorganism from a mixture of alamine 336 and oleic alcohol 1: 9. In the high-pressure reactor in which the reaction of the above-mentioned reaction formula 1 occurs, , Butyric acid was recovered as an extraction solvent layer at an efficiency of 74%.

Example  4. Butyric acid and Hexanoic acid  Used C8 - C12  Synthesis of compounds

Various C8-C12 compounds were synthesized from hexanoic acid and butyric acid using the various chemical catalysts and enzyme catalysts described above. That is, by carrying out a catalytic reaction and a hydrogenation reaction using hexanoic acid and butyric acid produced by caproic acid produsense galactitol riborans, butylbutyrate, hexylhexanoate, hexyl butyrate hexylbutyrate, butylhexanoate, hexanol, and butanol. For example, in the case of the synthesis of C8-C12 compounds using esterase, nobozyme 435 was treated with 1% butyric acid and 1% butanol, and after 60 minutes, more than 90% butyric acid and butanol were synthesized as butylbutyrate.

Example  5. Marine Biomass  And waste resources C4 - C6  Organic acid synthesis

Biomass, which can be easily obtained from the ocean, includes micro-algae. Because the microcrystals contain a large amount of galactose oligomers such as galactan that can be used as a substrate, caproic acid produsense galactitol riborans produces C4-C6 compounds using galactitol, which is a degradation product of the antler, , And the decomposition products of galactitol and the like were obtained from P. japonica using the following method.

First, the mugwort collected from the sea was pretreated by washing, drying, and grinding to try glycation. The dried beans were washed three times and then dried. The dried beads were pulverized using a blender, immersed in a solution containing 0.5 to 2% of H ₂ SO ₄ , acid treated and heated to 90 to 120 ° C for 0.5 to 1 hour , And then the properties were observed. Referring to FIG. 8, after the heat treatment, the mugwort, which was not treated with sulfuric acid, was easily gelated due to the effect of galactan. However, the mugwort pretreated with sulfuric acid was hydrolyzed and liquefied. Analysis of the liquefied solution by HPLC-ELSD showed that galactan was hydrolyzed to obtain several types of monosaccharides, among which galactose was the most abundant, accounting for 60 to 70% of the total. The resulting galactose was added with hydrogen under high pressure and high temperature conditions, and galactitol was prepared using a hydrogenation catalyst.

Name of depository: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM10991P

Payment date: 20090216

<110> Industry-University Cooperation Foundation Hanyang University <120> Novel strain having C4 and C6 organic acid producing capacity,          and method for producing bio-fuel using the strain <130> JKP-0051 <160> 1 <170> KoPatentin <210> 1 <211> 1259 <212> DNA <213> Unknown <220> <223> Caproiproducens galactitovorans <400> 1 gcgtgagtaa cctgcctttc agagggggat aacgtctgga aacggacgct aataccgcat 60 aacatttctg tgccgcatgg gatgggaatc aaaggaggaa tccgctgaga gatggactcg 120 cgtccgatta gctagttggt gagataaagg cccaccaagg cgacgatcgg tagccggact 180 gagaggttga acggccacat tgggactgag acacggccca gactcctacg ggaggcagca 240 gtgggggata ttgcacaatg gaggaaactc tgatgcagca acgccgcgtg agggaagaag 300 gtcttcggat tgtaaacctt tgtccttggt gacgaaaaga atgacggtag ccaaggagga 360 agctccggct aactacgtgc cagcagccgc ggtaatacgt agggagcaag cgttgtccgg 420 atttactggg tgtaaagggt gcgtaggcgg ctctgcaagt caggcgtgaa aaccatgggc 480 ttaacccatc ggattgcgtt tgaaactgtg gagcttgagt gaagtagagg taggcggaat 540 tcccggtgta gcggtgaaat gcgtagagat cgggaggaac accagtggcg aaggcggctt 600 actgggcttt aactgacgct gaggcacgaa agcatgggta gcaaacagga ttagataccc 660 tggtagtcca tgccgtaaac gatgattact aggtgtgggg ggtctgaccc cttccgtgcc 720 ggagttaaca caataagtaa tccacctggg gagtacggcc gcaaggttga aactcaaagg 780 aattgacggg ggcccgcaca agcagtggag tatgtggttt aattcgaagc aacgcgaaga 840 accttaccag gtcttgacat ccaactaacg aagcagagat gcattaggtg cccttcgggg 900 aaagttgaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 960 tcccgcaacg agcgcaaccc ttgtgattag ttgctacgca agagcactct aatcagactg 1020 ccgttgacaa aacggaggaa ggtggggacg acgtcaaatc atcatgccct ttatgacctg 1080 ggctacacac gtactacaat ggctgttaac agagggaagc aagcccgcga gggggagcaa 1140 aaccctaaaa acagtctcag ttcggatcgc aggctgaaac ccgcctgcgt gaagttggaa 1200 ttgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacac 1259

Claims (14)

Caprocytiludens galactitolivorans strain (KCCM 10991P), which has the ability to produce C4 organic acids and C6 organic acids simultaneously when cultivated in a carbon source medium. [2] The culture medium according to claim 1, wherein the carbon source medium comprises at least one saccharide selected from the group consisting of glycerol, glucose, galactitol, mannose, and fructose. The caprocycloprotein synthase of claim 1, wherein the C4 organic acid is butyric acid and the C6 organic acid is hexanoic acid. a) culturing a strain of caproicprodense galactibolans in a carbon source medium to produce a C4 organic acid and a C6 organic acid;
b) separating the C4 organic acid and C6 organic acid from the product of step a);
c) adding C4 alcohols and C6 alcohols having the same mole number as the C4 organic acids and C6 organic acids separated from step b) to prepare a mixture;
d) performing a catalytic reaction on the mixed solution of step c) to prepare a C8-C12 compound; And
e) producing a C4 alcohol and a C6 alcohol by reacting the C8-C12 compound prepared from step d) with hydrogen gas
&Lt; / RTI &gt; 5. The method of claim 4, wherein the carbon source medium comprises at least one saccharide selected from the group consisting of glycerol, glucose, galactitol, mannose, and fructose. 5. The method of claim 4, wherein the C4 organic acid is butyric acid and the C6 organic acid is hexanoic acid. 5. The method for producing a biofuel according to claim 4, wherein the cultivation in step a) is carried out by anaerobic culture at 35 DEG C to 45 DEG C for 24 to 72 hours. 5. The method according to claim 4, wherein the cultivation in step a) is carried out in a culture medium containing galactitol, fructose, mannose, tagatose, glucose and glycerol. 5. The method according to claim 4, wherein the separation in step b) is carried out by using an ion exchange resin or by extracting the C4 organic acid and C6 organic acid from the culture solution using an organic solvent immiscible with water. A method of manufacturing a fuel. 5. The method of claim 4, wherein the C4 alcohol is butanol, and the C6 alcohol is hexanol. 5. The method of claim 4, wherein the catalytic reaction in step d) is carried out in the presence of a catalyst selected from the group consisting of zeolite, heteropoly acids, silica-alumina, Nafion-H, p-toluenesulfonic acid acid), SO4 ^{2 -} / ZrO ₂ Or SO 4 ^{2 -} / TiO ₂ -La ₂ O ₃ super acid catalyst. 5. The method according to claim 4, wherein the catalytic reaction in step d) is carried out by adding at least one enzyme selected from the group consisting of Esterase and Lipase. 5. The method for producing a biofuel according to claim 4, wherein the hydrogen gas in step (e) is hydrogen gas generated from step (a). 5. The method according to claim 4, wherein the carbon source in step a) is prepared by physically pulverizing, washing and hydrolyzing biomass or organic waste.

Images